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A microarray analysis of developmentally regulated genes during macronuclear differentiation in the stichotrichous ciliate Stylonychia lemnae.

After sexual reproduction in ciliated protozoa a new macronucleus differentiates from a micronuclear derivative. In the course of macronuclear development dramatic DNA- and chromatin reorganisation processes occur, which include splicing of DNA sequences such as IES (internal eliminated sequences) and transposon-like elements during formation of polytene chromosomes, degradation of the polytene chromosomes and specific elimination of micronuclear-specific DNA, de novo addition of telomeres and specific amplification of DNA sequences. In order to understand the molecular basis of this nuclear differentiation process, analysis of developmentally regulated genes seems to be a necessary prerequisite. We performed a microarray analysis to identify genes differentially expressed during macronuclear differentiation. 467 sequences from cDNA libraries were identified as possible candidates from which 384 sequences were further characterised by sequence analysis. These sequences were identified, if possible, by DNA and protein BLAST analysis. Expression of one of these sequences was silenced by RNAi and a preliminary functional analysis performed. Results presented in this study provide the basis for a functional characterisation of genes differentially expressed during this nuclear differentiation process.

Animals↗

Transcriptional analysis of nucleolar dominance in polyploid plants: biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica.

Nucleolar dominance is an epigenetic phenomenon that describes the formation of nucleoli around rRNA genes inherited from only one parent in the progeny of an interspecific hybrid. Despite numerous cytogenetic studies, little is known about nucleolar dominance at the level of rRNA gene expression in plants. We used S1 nuclease protection and primer extension assays to define nucleolar dominance at a molecular level in the plant genus Brassica. rRNA transcription start sites were mapped in three diploids and in three allotetraploids (amphidiploids) and one allohexaploid species derived from these diploid progenitors. rRNA transcripts of only one progenitor were detected in vegetative tissues of each polyploid. Dominance was independent of maternal effect, ploidy, or rRNA gene dosage. Natural and newly synthesized amphidiploids yielded the same results, arguing against substantial evolutionary effects. The hypothesis that nucleolar dominance in plants is correlated with physical characteristics of rRNA gene intergenic spacers is not supported in Brassica. Furthermore, in Brassica napus, rRNA genes silenced in vegetative tissues were found to be expressed in all floral organs, including sepals and petals, arguing against the hypothesis that passage through meiosis is needed to reactivate suppressed genes. Instead, the transition of inflorescence to floral meristem appears to be a developmental stage when silenced genes can be derepressed.

Base Sequence↗

Expression patterns of developmental regulatory genes show comparable divisions in the telencephalon of Xenopus and mouse: insights into the evolution of the forebrain.

In this study, we review data on the existence of comparable divisions and subdivisions in the telencephalon of different groups of tetrapods based on expression of some developmental regulatory genes, having a particular focus in the comparison of the anuran amphibian Xenopus and the mouse. The available data on Xenopus, mouse, chick and turtle indicate that apparently all tetrapod groups possess the same molecularly distinct divisions and subdivisions in the telencephalon. This basic organization was likely present in the telencephalon of stem tetrapods. Each division/subdivision is characterized by expression of a unique combination of developmental regulatory genes, and appears to represent a self-regulated and topologically constant histogenetic brain compartment that gives rise to specific groups of cells. This interpretation has an important consequence for searching homologies, since a basic condition for cell groups in different vertebrates to be considered homologous is that they originate in the same compartment. However, evolution may allow individual cell groups derived from comparable (field homologous) subdivisions to be either similar or dissimilar across the vertebrate groups, giving rise to several possible scenarios of evolution, which include both the evolutionary conservation of similar (homologous) cells or the production of novel cell groups. Finally, available data in the lamprey, a jawless fish, suggest that not all telencephalic subdivisions were present at the origin of vertebrates, raising important questions about their evolution.

Animals↗

Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid- and hindbrain in a gene dose-dependent manner.

In vertebrates and insects, the homeobox transcription factors of the engrailed family have a dual function. They take part in regionalization during early embryogenesis and later in neuronal specification. In mammals, two engrailed homologues exist, engrailed-1 and engrailed-2, which are expressed in a broad band around the isthmus at an age when the serotonergic and noradrenergic neurons in mid/hindbrain are generated. The analysis of engrailed-1 and -2 double mutant mice revealed a specific, redundant, and gene dose-dependent requirement of the two transcription factors for the development of the serotonergic dorsal raphe nucleus and the noradrenergic locus caeruleus. Both nuclei are lost in engrailed double mutant mice; however, directly adjacent nuclei of the same neurotransmitter phenotype are not affected. An almost identical phenotype is found in mutant mice null for Wnt1, indicating that the engrailed genes provide essential positional information for the development of the two nuclei during early embryogenesis.

Animals↗

Primary sensory neurons express a Shaker-like potassium channel gene.

Developmentally regulated action potentials are a hallmark of Rohon-Beard cells, a class of sensory neurons. In these neurons as well as other primary spinal neurons of Xenopus laevis, the functional differentiation of delayed-rectifier potassium current regulates the waveform of the action potential during the initial day of its appearance. Later, the acquisition of another voltage-dependent potassium current--the A current--plays a major role in regulating excitability. In order to understand the molecular basis of this functional differentiation, genes encoding voltage-dependent potassium currents expressed in the embryonic amphibian nervous system are being cloned. Here, we report the functional properties and developmental localization of a second Xenopus Shaker-like gene (Xenopus Kv 1.1; XSha1; GenBank accession number M94258) encoding a potassium current. Homology screening with the mouse gene MBK1 led to its isolation. Functional expression in oocytes identifies it as a delayed-rectifier current when assembled as a homooligomeric structure. Specific transcripts corresponding to XSha1 and to the previously cloned gene XSha2 are both detectable by RNase protection in RNA isolated from the embryonic nervous system. However, whole-mount in situ hybridization reveals the temporal pattern and cellular localization of XSha1 but not XSha2 mRNA, suggesting that the concentration of XSha2 transcripts in individual cells is lower than the threshold for detection by this method. Of particular interest, Rohon-Beard cells express XSha1 mRNA. In addition, XSha1 mRNA is detected in several structures containing neural crest derivatives including spinal ganglia, the trigeminal ganglion, and branchial arches; its presence in motor nerves and lateral spinal tracts suggests that both CNS and PNS glia express the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Binding of thymic factors to the conserved decanucleotide promoter element of the T-cell receptor V beta gene is developmentally regulated and is absent in SCID mice.

The gene segments encoding the beta chain of the T-cell antigen receptor undergo rearrangement in a precise developmental order: a D beta gene segment joins to a J beta gene segment prior to the rearrangement of a V beta gene segment to join the D/J beta fusion. Current evidence suggests that the rearrangement of V beta is restricted to T cells, whereas D-to-J beta rearrangements may occur in both B and T cells. Thus, the T-cell specificity seems to be regulated by the V beta coding region or its 5' flanking sequence. In support of this hypothesis, evidence is provided for thymus-specific factors that bind a highly conserved 10-base-pair (decamer) sequence that is an essential promoter element in mouse and human V beta genes. The presence of decamer-binding activities was assayed by gel mobility-shift analysis using protein extracts from thymus, spleen, and nonlymphoid organs of adult mice. Two shifted complexes, designated T2 and T3, were seen only when the decamer was incubated with extracts from thymus. When extracts from mice of various gestational ages were tested for decamer-binding activity, one of the thymus-specific complexes, T2, was first detected at day 16; this coincides with the time of initial activation of the V beta locus. No decamer-binding activity was detected in extracts prepared from the thymuses of SCID (severe combined immunodeficiency) mice, which characteristically fail to rearrange these genes. Moreover, neither T2 nor T3 was detectable with extracts from spleen or from two T-cell lines that express the beta chain; this suggests that the presence of these two complexes is not absolutely required for transcription of the T-cell receptor beta locus. We conclude that there are tissue-specific and developmentally regulated factors that form complexes with the decamer sequence 5' of V beta; these may represent initiation factors that control the activation of germ-line T-cell receptor V beta genes for transcription and/or rearrangement.

Age Factors↗

Zebrafish Hoxa and Evx-2 genes: cloning, developmental expression and implications for the functional evolution of posterior Hox genes.

Vertebrate Hox genes are required for the establishment of regional identities along body axes. This gene family is strongly conserved among vertebrates, even in bony fish which display less complex ranges of axial morphologies. We have analysed the structural organization and expression of Abd-B related zebrafish HoxA cluster genes (Hoxa-9, Hoxa-10, Hoxa-11 and Hoxa-13) as well as of Evx-2, a gene closely linked to the HoxD complex. We show that the genomic organization of Hoxa genes in fish resembles that of tetrapods albeit intergenic distances are shorter. During development of the fish trunk, Hoxa genes are coordinately expressed, whereas in pectoral fins, they display transcript domains similar to those observed in developing tetrapod limbs. Likewise, the Evx-2 gene seems to respond to both Hox- and Evx-types of regulation. During fin development, this latter gene is expressed as the neighbouring Hox genes, in contrast to its expression in the central nervous system which does not comply with colinearity and extends up to anterior parts of the brain. These results are discussed in the context of the functional evolution of Hoxa versus Hoxd genes and their different roles in building up paired appendages.

Amino Acid Sequence↗

Expression of a targeted lambda 1 light chain gene is developmentally regulated and independent of Ig kappa rearrangements.

Immunoglobulin light chain (IgL) rearrangements occur more frequently at Ig kappa than at Ig lambda. Previous results suggested that the unrearranged Ig kappa locus negatively regulates Ig lambda transcription and/or rearrangement. Here, we demonstrate that expression of a VJ lambda 1-joint inserted into its physiological position in the Ig lambda locus is independent of Ig kappa rearrangements. Expression of the inserted VJ lambda 1 gene segment is developmentally controlled like that of a VJ kappa-joint inserted into the Ig kappa locus and furthermore coincides developmentally with the occurrence of Ig kappa rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Ig lambda locus occurs in the presence of an unrearranged Ig kappa locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in approximately 30% of lambda 1 expressing B cell progenitors.

Animals↗

Characterization of a stress-induced, developmentally regulated gene family from soybean.

We describe a family of stress-induced, developmentally regulated soybean genes for which cDNAs have been obtained from two different cultivars (Glycine max cv. Mandarin and Glycine max cv. Williams). The mRNAs corresponding to these cDNAs, called SAM22 and H4, respectively, accumulate predominantly in the roots of soybean seedlings but are present at high levels in the roots and leaves of mature plants. SAM22 accumulation is especially dramatic in senescent leaves. In addition, SAM22 accumulation can be induced on young leaves by wounding or by transpiration-mediated uptake of salicylic acid, methyl viologen, fungal elicitor, hydrogen peroxide or sodium phosphate (pH 6.9). Taken together, these data indicate that the genes corresponding to SAM22 and H4 are induced by various stresses and developmental cues. Southern blot analysis indicates that multiple copies of sequences related to SAM22 exist in the soybean genome. We also show that the nucleotide sequences of the cDNAs corresponding to SAM22 and H4 are 86% identical at the nucleotide level to each other and 70% identical at the amino acid level to the 'disease resistance response proteins' of Pisum sativum.

Amino Acid Sequence↗

The Arabidopsis extensin gene is developmentally regulated, is induced by wounding, methyl jasmonate, abscisic and salicylic acid, and codes for a protein with unusual motifs.

A single-copy extensin gene (atExt1) has been isolated from Arabidopsis thaliana (L.) Heynh. The deduced amino acid sequence consists of 374 amino acids which are organised into highly ordered repeating blocks in which Ser(Pro)4 and Ser(Pro)3 motifs alternate. Two copies of the Tyr-X-Tyr-Lys motif and 13 copies of the Val-Tyr-Lys motif are present, showing that this extensin may be highly cross-linked, possessing the capacity for both intra and inter-molecular bond formation. The gene atExt1 is normally expressed in the root and is silent in the leaf; wounding reverses this pattern, turning on the gene in the leaf and repressing it in the root. The promoter contains motifs which have been found to activate plant defence genes in response to salicylic acid, abscisic acid and methyl jasmonate; when these compounds are applied to the roots, the atExt1 gene is activated in the leaf.

Abscisic Acid↗

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6. 5-27 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatio-temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.

Adenocarcinoma↗

Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos.

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.

Actins↗

A family-based association study does not support DYX1C1 on 15q21.3 as a candidate gene in developmental dyslexia.

We applied a family-based association approach to investigate the role of the DYX1C1 gene on chromosome 15q as a candidate gene for developmental dyslexia (DD) to 158 families containing at least one dyslexic child. We directly sequenced exons 2 and 10 of the DYX1C1 gene and found eight single nucleotide polymorphism (SNPs), three of which (-3G>A, 1249 G>T, 1259 C>G) were suitable for the genetic analyses. We performed single- and multimarker association analyses with DD as a categorical trait by FBAT version 1.4 and TRANSMIT version 2.5.4 programs. Our sample had a power of at least 80% to detect an association between the selected phenotypes and the informative polymorphisms at a significance level of 5%. The results of the categorical analyses did not support the involvement of the DYX1C1 gene variants in this sample of dyslexics and their relatives. Quantitative and multimarker analyses, which provide greater power to detect loci with a minor effect, consistently yielded nonsignificant results. While D1X1C1 is a good candidate gene for DD, we were unable to replicate the original findings between DYX1C1 gene and DD, perhaps due to genetic heterogeneity.

Adolescent↗

Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain.

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.

Animals↗

Identification of a developmentally regulated gene for a 140-kDa secretory protein in salivary glands of Chironomus tentans larvae.

Secretory proteins are synthesized in salivary glands of the insect, Chironomus tentans, and assemble in vivo into silk-like threads which aquatic larvae use to construct tubes for filter feeding and pupation. Thus far, all known secretory protein genes contain repetitious protein-coding sequences and are located in cytological structures known as Balbiani rings, giant puffs found on polytene secretory cell chromosomes. In this paper we describe the identification of another secretory protein gene which is comprised of repeated sequences; however, this gene is not located in a Balbiani ring. Two partial cDNA clones from a 3.6-kilobase pair poly(A)+ RNA were sequenced and found to contain two open reading frames for protein synthesis. Antibodies were raised against synthetic oligopeptides whose sequences were derived from these two open reading frames. An immunoaffinity-purified antibody for one of these peptides bound specifically to a 140-kDa secretory protein (sp140). The cDNA sequences contain tandem repeats of 42 base pairs which encode a repeat of 14 amino acids with a composition and oligopeptide sequence similar to other secretory proteins. The C. tentans genome contains about 70 copies of this 42-base pair repeat organized as a contiguous block of 3 kilobase pairs or less. The sp140 gene was mapped by in situ hybridization to polytene chromosome band I-17-B. Developmental studies of protein accumulation, steady-state levels of mRNA, and relative transcription rate suggested that the sp140 gene is developmentally regulated so that maximal expression is achieved during the prepupal stages of the fourth larval instar. Based upon these results we proposed that sp140 gene belongs to a prepupal class of secretory protein genes. While the sp140 gene shares structural and expression characteristics with other secretory protein genes, its unique chromosomal location shows that this multigene family is not restricted to Balbiani rings.

Amino Acid Sequence↗

Developmental regulation of elastin gene expression.

Developmental regulation of elastin gene expression appears to be exerted primarily at the transcriptional level. Although the elastin gene promoter possesses features of "housekeeping" genes, these characteristics do not preclude transcriptional regulation as shown with a number of other gene promoters of this class. Direct evidence for transcriptional regulation has been obtained by nuclear run-on analysis of nuclei isolated from developing lung and aortic tissues and indirectly through elastin promoter activity in transgenic mice and transient tissue transfections of embryonic lung and aortic tissues. Although several different modulators have been proposed to control the developmental activation of elastin gene expression, only insulin-like growth factor I has been experimentally linked to increased transcription by in vivo studies. This link is specific for aortic smooth muscle cells in which cell cycle control appears intimately associated with elastogenesis. Recent studies suggest that progress in understanding developmental activation of the elastin gene lies in transgenic models and organ transfection assays that assess the direct relevancy of the modulators and cis- and trans-acting factors involved.

Animals↗

Stage and developmental specific gene expression during mammalian spermatogenesis.

Spermatogenesis is a complex developmental process which involves amplification of germinal stem cells, their differentiation into spermatocytes, meiotic division and finally transformation into mature spermatozoa. Therefore, spermatogenesis provides an interesting system for examining the regulation of gene expression during development and differentiation. The genes expressed during spermatogenesis can be divided into two main groups: diploid and haploid expressed genes. In this review, we report about the regulation of expression of a diploid expressed gene, namely the proacrosin gene, and that of a haploid expressed gene, the transition protein 2 gene.

Acrosin↗

Modular cis-regulatory organization of developmentally expressed genes: two genes transcribed territorially in the sea urchin embryo, and additional examples.

The cis-regulatory systems that control developmental expression of two sea urchin genes have been subjected to detailed functional analysis. Both systems are modular in organization: specific, separable fragments of the cis-regulatory DNA each containing multiple transcription factor target sites execute particular regulatory subfunctions when associated with reporter genes and introduced into the embryo. The studies summarized here were carried out on the CyIIIa gene, expressed in the embryonic aboral ectoderm and on the Endo16 gene, expressed in the embryonic vegetal plate, archenteron, and then midgut. The regulatory systems of both genes include modules that control particular aspects of temporal and spatial expression, and in both the territorial boundaries of expression depend on a combination of negative and positive functions. In both genes different regulatory modules control early and late embryonic expression. Modular cis-regulatory organization is widespread in developmentally regulated genes, and we present a tabular summary that includes many examples from mouse and Drosophila. We regard cis-regulatory modules as units of developmental transcription control, and also of evolution, in the assembly of transcription control systems.

Actins↗